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Illustration of the fluorescent ratio probe (OPDEA/BOD-NO2) with hypoxia targeting and hypoxia activation capacity for in vivo tumour imaging.

The current trend in industrial biotechnology is to move from batch or fed‐batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady‐state conditions and explored the utility of promoters of these genes (Chr1–4_0586 and FragB_0052) in optimizing the expression of two different r‐proteins, human lysozyme (HuLy), and the anti‐idiotypic antibody fragment, Fab‐3H6, in comparison with the widely used glyceraldehyde‐3‐phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome‐scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab‐3H6. These results showed that a genome‐scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r‐protein and then this information can be used to tailor a cultivation medium to increase production.

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc.

A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

The preparation, characterisation and testing of stable non-porous coated perfluorocarbon supports functionalised with the metal chelate, iminodiacetic acid (IDA) is described. Polyvinyl alcohol (PVA), a neutral hydrophilic polymer was esterified with perfluorooctanoyl chloride and anchored to the surface of solid perfluorocarbon particles through multiple fluorophilic interactions. The PVA-coated particles were then activated by epoxidation and coupled with IDA. The presence of surface-attached chelates was clearly demonstrated by the binding and selective desorption of Zn2+ ions. Three particulate perfluorocarbons were selected as potential starting materials and the conditions for preparation of metal chelating adsorbents optimised with respect to ease of manufacture, ligand density and binding capacity towards a monoclonal antibody known to bind to commercial Zn(2+)-IDA supports. The choice of base particle strongly influenced the ligand densities and specific binding capacities towards the monoclonal antibody that could be achieved under optimal preparative conditions. Possible ways in which these metal chelating adsorbents may be employed to recover the monoclonal antibody directly from culture vessels are discussed.

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.

Ground state antimony atoms, Sb[5p3(4S)], have been studied by time-resolved resonance fluorescence at λ= 231.1 nm (64P½→ 54S) in the “single-shot” mode. The antimony atoms were generated by pulsed irradiation of SbMe3 using a “magnetically-pinched”, Garton light source with a high spectral output in the vacuum ultraviolet. Optical excitation of the resulting Sb(54S) was effected by means of a sealed microwave-powered atomic emission source, and the resonance fluorescence was monitored photoelectrically with a photomultiplier tube gated with a “pre-trigger” system. Kinetic studies of Sb(54S) in the presence of NO and of O2 are described and the resulting rate constants are compared with analogous data derived previously using time-resolved resonance absorption at the same wavelength, and discussed in terms of departures from the standard Beer–Lambert law for light absorption. Radiation trapping calculations are also described which involve summation of the Voigt profiles over all the individual nuclear hyperfine components that contribute to the λ= 231.1 nm transition. Solutions of the diffusion equation for radiation with different boundary conditions are employed in the calculations of the relationship between fluorescence intensity and particle density. The results are compared with the experimental fluorescence intensity calibration for this system.

We present a kinetic study of ground state arsenic atoms, generated photochemically and monitored by fluorescence in the time-resolved mode following optical excitation. As[4p3(4S)] was derived from the repetitive pulsed irradiation of low pressures of either AsMe3 or AsCl3, in the presence of excess helium, in a flow system which is kinetically equivalent to a static system. The transient atoms were then monitored photoelectrically by summation of the optically excited fluorescence, principally at the wavelengths λ= 189.04, 193.76 and 197.76 and 197.23 nm [4p25s(4P5/2, , ½)→ 4p3(4S), respectively] using a “pre-trigger” photomultiplier gating system coupled with singal averaging. The decay of the arsenic atoms in the presence of the added reactant gases O2, Cl2, NO and C2H4 is described, for which second-order reaction rate constants are reported. These data are compared with previous results derived from time-resolved resonance absorption studies at λ= 189.04 nm. The two sets of rate constants resulting from the resonance absorption fluorescence and absorption studies are then employed in order to discuss the modified Beer–Lambert Law, Itr=I0 exp [–Iµ(cl)γ], that has been used in the resonance absorption measurements. Finally, we present a model radiation trapping calculation including the effects of nuclear hyperfine interaction using the diffusion theory of radiation for the three wavelengths monitored in fluorescence, and calculate the relationship between fluorescence intensity and particle density. This is compared with empirical measurements of the fluorescence intensity against particle density calibration.

The temporal behaviour of ground state phosphorus atoms, P[3p3(4S)], generated by repetitive pulsed irradiation in a flow system, has been studied by time-resolved resonance fluorescence. P(34S) was generated photochemically from PCI3 in the presence of excess helium buffer gas and optically excited by means of the emission from a microwave-powered atomic spectroscopic source. The total resonance fluorescence in the vacuum ultraviolet from the systems P[3p24s(4P½,,5/2)→ 3p3(4S)](λ= 178.77, 178.29 and 177.50 nm, respectively) and P[3p4(4P5/2,,½)→ 3p3(4S)](λ= 167.97, 167.46 and 167.17 nm, respectively) was monitored photoelectrically and the signal-averaged results employed to characterise the kinetic decay of the phosphorus atom in the presence of various added gases. An empirical calibration of the relationship between the fluorescence intensity and the relative particle density of P(34S) was established and the linear region employed for kinetic study. Second-order rate constants (kR, cm3 molecule–1 s–1, 300 K) are reported for the reaction of P(34S) with the following gases: O2(2.1 ± 0.3 × 10–12), Cl2(3.4 ± 0.3 × 10–12), NO (5.3 ± 0.5 × 10–13), C2H2(1.2 ± 0.1 × 10–13), C2H4(6.8 ± 0.8 × 10–13) and PCl3(1.7 ± 0.3 × 10–12). These data are compared with previous results obtained by time-resolved resonance line absorption and used to consider further the modified Beer–Lambert law, Itr=I0 exp [–Iµ(cl)γ] used in the resonance absorption measurements. Finally, radiation trapping calculations are described for the systems P[3p24s(4PJ)] and P[3p4(4PJ)]→ P[(34S)] using the diffusion theory of radiation. Line shapes employing the summation of Voigt profiles for all the nuclear hyperfine components contributing to a given electronic transition are used. The calculated relationships between the fluorescence intensity and P[(34S)] for various boundary conditions of the diffusion equation are compared with the empirical calibration.

Ground state nitrogen atoms, N(24S), generated by pulsed photochemical initiation, have been, studied by time-resolved resonance fluorescence following optical excitation in the vacuum ultraviolet, λ= 120 nm, N[2p,23s(4P½,,[graphic omitted])→ 2p3(4S)]. Atomic nitrogen, principally in the optically metastable doublet states, N[2p3(2DJ, 2PJ)], was produced by the repetitive vacuum ultraviolet photolysis of N2O and collisionally quenched to the 4S state by the use of N2 as a buffer gas. Photoelectric detection of the vacuum u.v. signals employed photon counting coupled with signal summation in a multiscalar. Considerable care was required to eliminate spurious photon signals at λ= 130 nm due to O(23PJ)[O(33S1)→ O(23PJ)]. Absolute rate data for the collisional removal of N(24S) in the presence of the gases NO, NO2, O3 and C2H4 are presented and compared with previously reported data. The results for N + C2H4 are further discussed in relation to the “HCN limiting yield” method that has been employed hitherto as a marker for N atom kinetics.

We describe a kinetic investigation of H and D atoms in their electronic ground states by the method of time-resolved Lyman-α resonance fluorescence in the vacuum ultraviolet following pulsed photochemical generation of the atoms. The method involved repetitive pulsing, photon counting and signal averaging. Absolute rate constants (k/cm3 molecule–1 s–1, 300 K) are reported for the following reactants; H + H2S, (8.6 ± 0.5)× 10–13; D + D2S, (7.9 ± 0.4)× 10–13; H + HBr, (6.0 ± 0.1)× 10–12; D + HBr, (4.1 ± 0.1)× 10–12; H + DBr, (4.7 ± 0.1)× 10–12; D + DBr, (2.7 ± 0.1)× 10–12. The rates for the reactions with HBr and DBr are compared with values previously derived from e.p.r. measurements on H and D in a flow discharge system.

Cornea diseases are a leading cause of blindness and the disease burden is exacerbated by the increasing shortage around the world for cadaveric donor corneas. Despite the advances in the field of regenerative medicine, successful transplantation of laboratory‐made artificial corneas is not fully realized in clinical practice. The causes of failure of such artificial corneal implants are multifactorial and include latent infections from viruses and other microbes, enzyme overexpression, implant degradation, extrusion or delayed epithelial regeneration. Therefore, there is an urgent unmet need for developing customized corneal implants to suit the host environment and counter the effects of inflammation or infection, which are able to track early signs of implant failure in situ. This work reports a nanotoolbox comprising tools for protection from infection, promotion of regeneration, and noninvasive monitoring of the in situ corneal environment. These nanosystems can be incorporated within pro‐regenerative biosynthetic implants, transforming them into theranostic devices, which are able to respond to biological changes following implantation. Funding agencies: EU H2020 Marie Sklodowska-Curie Individual Fellowship [706694]; MIIC Strategic Postdoc Grant; MIIC Seed Grant at Linkoping University (LiU), Sweden